Novel compounds

ABSTRACT

The invention provides ratB polypeptides and DNA (RNA) encoding ratB polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing ratB polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

[0001] This application claims benefit of U.S. 60/037,857 filed Feb. 7,1997; U.S. 60/044,365 filed Apr. 28, 1997; U.S. 60/044,366 filed Apr.28, 1997 and U.S. 60/045,129 filed Apr. 28, 1997.

FIELD OF THE INVENTION

[0002] This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the rat family, hereinafter referred to as “ratB”.

BACKGROUND OF THE INVENTION

[0003] It is particularly preferred to employ Staphylococcal genes andgene products as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. aureus is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thrombophlebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome.

[0004] The frequency of Staphylococcus aureus infections has risendramatically in the past 20 years. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Staphylococcus aureus strains which are resistant tosome or all of the standard antibiotics. This has created a demand forboth new anti-microbial agents and diagnostic tests for this organism.

[0005] Aminoacyl-tRNA synthetases (aaRS) catalyse the ligation of aminoacids to their cognate tRNA species in all cellular organisms. Ingeneral, each of the twenty amino acids that are incorporated intogrowing polypeptide chains has a corresponding aaRS. However, it is nowwell documented that this is not universally true and thatglutaminyl-tRNA synthetase (QRS) activity is absent in all Gram-positiveprokaryotes examined, in some Gram-negative prokaryotes and in theplastids of some, and possibly all, eukaryotes. Despite the absence ofglutaminyl-tRNA synthetase activity, cells are clearly able to producethe Gln-tRNAGln required for accurate protein synthesis. The mechanismby which this is achieved involves the formation of Glu-tRNAGln as anintermediate that is produced by the misaminoacylation of tRNAGln byglutamyl-tRNA synthetase (ERS). The ‘correct’ end product, Gln-tRNAGln,is formed from Glu-tRNAGln by transfer of an amine group to the ligatedglutamate residue. This reaction is catalysed by a tRNA- andMg2+/ATP-dependent amidotransferase. (RNA-dependentAmidoTransferase—RAT). Inhibition of this apparently ubiquitous reactionin Gram-positive organisms, and some Gram-negative organisms, wouldeffectively lead to Gln-tRNAGln starvation and to the synthesis ofaberrant proteins and the consequent cessation of bacterial proteinsynthesis.

[0006] Clearly, there is a need for factors, such as the novel compoundsof the invention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists which can play a role inpreventing, ameliorating or correcting infections, dysfunctions ordiseases.

[0007] The polypeptides of the invention have amino acid sequencehomology to a known Bacillus subtilis PET112-like protein, encoded bypart of the DNA sequence from GenBank deposition with accession numberU49790 protein.

SUMMARY OF THE INVENTION

[0008] It is an object of the invention to provide polypeptides thathave been identified as novel ratB polypeptides by homology between theamino acid sequence set out in Table 1 [SEQ ID NO: 2] and a known aminoacid sequence or sequences of other proteins such as Bacillus subtilisPET112-like protein, encoded by part of the DNA sequence from GenBankdeposition with accession number U49790 protein.

[0009] It is a further object of the invention to providepolynucleotides that encode ratB polypeptides, particularlypolynucleotides that encode the polypeptide herein designated ratB.

[0010] In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding ratB polypeptides comprisingthe sequence set out in Table 1 [SEQ ID NO:1] which includes a fulllength gene, or a variant thereof.

[0011] In another particularly preferred embodiment of the inventionthere is a novel ratB protein from Staphylococcus aureus comprising theamino acid sequence of Table 1 [SEQ ID NO:2], or a variant thereof.

[0012] In accordance with another aspect of the invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressible by the Staphylococcus aureus WCUH 29 strain contained in thedeposited strain.

[0013] A further aspect of the invention there are provided isolatednucleic acid molecules encoding ratB, particularly Staphylococcus aureusratB, including mRNAs, cDNAs, genomic DNAs. Further embodiment of theinvention include biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

[0014] In accordance with another aspect of the invention, there isprovided the use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of ratB and polypeptides encoded thereby.

[0015] Another aspect of the invention there are provided novelpolypeptides of Staphylococcus aureus referred to herein as ratB as wellas biologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

[0016] Among the particularly preferred embodiments of the invention arevariants of ratB polypeptide encoded by naturally occurring alleles ofthe ratB gene.

[0017] In a preferred embodiment of the invention there are providedmethods for producing the aforementioned ratB polypeptides.

[0018] In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

[0019] In accordance with certain preferred embodiments of theinvention, there are provided products, compositions and methods forassessing ratB expression, treating disease, for example, disease, suchas, infections of the upper respiratory tract (e.g., otitis media,bacterial tracheitis, acute epiglottitis, thyroiditis), lowerrespiratory (e.g., empyema, lung abscess), cardiac (e.g., infectiveendocarditis), gastrointestinal (e.g., secretory diarrhoea, splenicabsces, retroperitoneal abscess), CNS (e.g., cerebral abscess), eye(e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis,preseptal and orbital cellulitis, darcryocystitis), kidney and urinarytract (e.g., epididymitis, intrarenal and perinephric absces, toxicshock syndrome), skin (e.g., impetigo, folliculitis, cutaneousabscesses, cellulitis, wound infection, bacterial myositis) bone andjoint (e.g., septic arthritis, osteomyclitis), assaying geneticvariation, and administering a ratB polypeptide or polynucleotide to anorganism to raise an immunological response against a bacteria,especially a Staphylococcus aureus bacteria.

[0020] In accordance with certain preferred embodiments of this andother aspects of the invention there are provided polynucleotides thathybridize to ratB polynucleotide sequences, particularly under stringentconditions.

[0021] In certain preferred embodiments of the invention there areprovided antibodies against ratB polypeptides.

[0022] In other embodiments of the invention there are provided methodsfor identifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

[0023] In accordance with yet another aspect of the invention, there areprovided ratB agonists and antagonists, preferably bacteriostatic orbacteriocidal agonists and antagonists.

[0024] In a further aspect of the invention there are providedcompositions comprising a ratB polynucleotide or a ratB polpypeptide foradministration to a cell or to a multicellular organism.

[0025] Various changes and modifications within the spirit and scope ofthe disclosed invention will become readily apparent to those skilled inthe art from reading the following descriptions and from reading theother parts of the present disclosure.

[0026] GLOSSARY

[0027] The following definitions are provided to facilitateunderstanding of certain terms used frequently herein.

[0028] “Host cell” is a cell which has been transformed or transfected,or is capable of transformation or transfection by an exogenouspolynucleotide sequence.

[0029] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

[0030] Preferred parameters for polypeptide sequence comparison includethe following:

[0031] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970)

[0032] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci. USA. 89:10915-10919 (1992)

[0033] Gap Penalty: 12

[0034] Gap Length Penalty: 4

[0035] A program useful with these parameters is publicly available asthe “gap” program from Genetics Computer Group, Madison Wis. Theaformentioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

[0036] Preferred parameters for polynucleotide comparison include thefollowing:

[0037] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970)

[0038] Comparison matrix: matches=+10, mismatch=0

[0039] Gap Penalty: 50

[0040] Gap Length Penalty: 3

[0041] Available as: The “gap” program from Genetics Computer Group,Madison Wis. These are the default parameters for nucleic acidcomparisons.

[0042] Preferred polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide having at least a 50, 60, 70,80, 85, 90, 95, 97 or 100% identity to a polynucleotide referencesequence of SEQ ID NO:1, wherein said reference sequence may beidentical to the sequence of SEQ ID NO: 1 or may include up to a certaininteger number of nucleotide alterations as compared to the referencesequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO:1 by thenumerical percent of the respective percent identity and subtractingthat product from said total number of nucleotides in SEQ ID NO:1, or:

n_(n)≦x_(n)−(x_(n)·y),

[0043] wherein n_(n) is the number of nucleotide alterations, x_(n) isthe total number of nucleotides in SEQ ID NO:1, and y is 0.50 for 50%,0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%,0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and wherein any non-integerproduct of x_(n) and y is rounded down to the nearest integer prior tosubtracting is from x_(n). Alterations of a polynucleotide sequenceencoding the polypeptide of SEQ ID NO:2 may create nonsense, mis-senseor frameshift mutations in this coding sequence and thereby alter thepolypeptide encoded by the polynucleotide following such alterations.

[0044] Preferred polypeptide embodiments further include an isolatedpolypeptide comprising a polypeptide having at least a 50, 60, 70, 80,85, 90, 95, 97 or 100% identity to a polypeptide reference sequence ofSEQ ID NO:2, wherein said reference sequence may be identical to thesequence of SEQ ID NO: 2 or may include up to a certain integer numberof amino acid alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least oneamino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carbxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the aminno acids inthe reference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ ID NO:2by the numerical percent of the respective percent identity andsubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n_(a)≦x_(a)−(x_(a)·y),

[0045] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO:2, and y is 0.50 for 50%,0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%,0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and wherein any non-integerproduct of x_(a) and y is rounded down to the nearest integer prior tosubtracting it from x_(a).

[0046] “Isolated” means altered “by the hand of man” from its naturalstate, i.e., if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein.

[0047] “Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

[0048] “Polypeptide(s)” refers to any peptide or protein comprising twoor more amino acids joined to each other by peptide bonds or modifiedpeptide bonds. “Polypeptide(s)” refers to both short chains, commonlyreferred to as peptides, oligopeptides and oligomers and to longerchains generally referred to as proteins. Polypeptides may contain aminoacids other than the 20 gene encoded amino acids. “Polypeptide(s)”include those modified either by natural processes, such as processingand other post-translational modifications, but also by chemicalmodification techniques. Such modifications are well described in basictexts and in more detailed monographs, as well as in a voluminousresearch literature, and they are well known to those of skill in theart. It will be appreciated that the same type of modification may bepresent in the same or varying degree at several sites in a givenpolypeptide. Also, a given polypeptide may contain many types ofmodifications. Modifications can occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains, and theamino or carboxyl termini. Modifications include, for example,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a homo moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation, selenoylation, sulfation, transfer-RNA mediatedaddition of amino acids to proteins, such as arginylation, andubiquitination. See, for instance, PROTEINS-STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993) and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al, Meth. Enzymol. 182:626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann,N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched orcyclic, with or without branching. Cyclic, branched and branchedcircular polypeptides may result from post-translational naturalprocesses and may be made by entirely synthetic methods, as well.

[0049] “Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

[0050] The invention relates to novel ratB polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a novel ratB ofStaphylococcus aureus, which is related by amino acid sequence homologyto Bacillus subtilis PET112-like protein, encoded by part of the DNAsequence from GenBank deposition with accession number U49790polypeptide. The invention relates especially to ratB having thenucleotide and amino acid sequences set out in Table 1 [SEQ ID NO: 1]and Table 1 [SEQ ID NO: 2] respectively, and to the ratB nucleotidesequences of the DNA in the deposited strain and amino acid sequencesencoded thereby. TABLE 1 ratB Polynucleotide and Polypeptide Sequences(A) Sequences from Staphyloxoxxus aureus ratB polynucleotide sequence[SEQ ID NO:1]. 5′-ATGCATTTTGAAACAGTTATAGGACTTGAAGTTCACGTAGAGTTAAAAACGGACTCAAAAATGTTTTCTCCATCACCAGCGCATTTTGGAGCAGAACCTAACTCAAATACAAATGTTATCGACTTAGCATATCCAGGTGTCTTACCAGTTGTTAATAAGCGTGCAGTAGACTGGGCAATGCGTGCTGCAATGGCACTAAATATGGAAATCGCAACAGAATCTAAGTTTGACCGTAAGAACTATTTCTATCCAGATAATCCAAAAGCATATCAAATTTCTCAATTTGATCAACCAATTGGTGAAAATGGATATATCGATATCGAAGTCGACGGTGAAACAAAACGAATCGGTATTACTCGTCTTCACATGGAAGAAGATGCTGGTAAGTCAACACATAAAGGTGAGTATTCATTAGTTGACTTGAACCGTCAAGGTACACCGCTAATTGAAATCGTATCTGAACCAGATATTCGTTCACCTAAAGAAGCATATGCATATTTAGAAAAATTACGTTCAATTATTCAATACACTGGTGTATCAGACGTTAAGATGGAAGAGGGATCTTTACGTTGTGATGCTAACATCTCTTTGCGTCCATATGGTCAAGAAAAATTTGGTACTAAAGCCGAATTGAAAAACTTAAACTCATTTAACTATGTACGTAAAGGTTTAGAATATGAAGAAAAACGCCAAGAAGAAGAATTGTTAAATGGTGGAGAAATCGGACAAGAAACACGTCGATTTGATGAATCTACAGGTAAAACAATTTTAATGCGTGTTAAAGAAGGTTCTGATGATTACCGTTACTTCCCAGAGCCTGACATTGTACCTTTATATATTGATGATGCTTGGAAAGAGCGTGTTCGTCAGACAATTCCTGAATTACCAGATGAGCGTAAGGCTAAGTATGTAAATGAATTAGGTTTACCTGCATACGATGCACACGTATTAACATTGACTAAAGAAATGTCAGATTTCTTTGAATCAACAATTGAACACGGTGCAGATGTTAAATTAACATCTAACTGGTTAATGGGTGGCGTAAACGAATATTTAAATAAAAATCAAGTAGAATTATTAGATACTAAATTAACACCAGAAAATTTAGCAGGTATGATTAAACTTATCGAAGACGGAACAATGAGCAGTAAAATTGCGAAGAAAGTCTTCCCAGAGTTAGCAGCTAAAGGTGGTAATGCTAAACAGATTATGGAAGATAATGGCTTAGTTCAAATTTCTGATGAAGCAACACTTCTAAAATTTGTAAATGAAGCATTAGACAATAACGAACAATCAGTTGAAGATTACAAAAATGGTAAAGGCAAAGCTATGGGCTTCTTAGTTGGTCAAATTATGAAAGCGTCTAAAGGTCAAGCTAATCCACAATTAGTAAATCAACTATTAAAACAAGAATTAGATAAAAGA-3′ (B) ratB polypeptide sequence deduced fromthe polynucleotide sequence in this table [SEQ ID NO:2]. NH₂-MHFETVIGLEVHVELKTDSKMFSPSPAHFGAEPNSNTNVIDLAYPGVLPVVNKRAVDWAMRAAMALNMEIATESKFDRKNYFYPDNPKAYQISQFDQPIGENGYIDIEVDGETKRIGITRLHMEEDAGKSTHKGEYSLVDLNRQGTPLIEIVSEPDIRSPKEAYAYLEKLRSIIQYTGVSDVKMEEGSLRCDANISLRPYGQEKFGTKAELKNLNSPNYVRKGLEYEEKRQEEELLNGGEIGQETRRFDESTGKTILMRVKEGSDDYRYFPEPDIVPLYIDDAWKERVRQTIPELPDERKAKYVNELGLPAYDAHVLTLTKEMSDFFESTIEHGADVKLTSNWLMGGVNEYLNKNQVELLDTKLTPENLAGMIKLIEDGTMSSKIAKKVFPELAAKGGNAKQIMEDNGLVQISDEATLLKFVNEALDNNEQSVEDYKNGKGKAMGFLVGQIMKASKGQANPQLVNQLLKQELDKR-COOH (C)Polynucleotide sequence embodiments [SEQ ID NO:1].X-(R₁)_(n)-ATGCATTTTGAAACAGTTATAGGACTTGAAGTTCACGTAGAGTTAAAAACGGACTCAAAAATGTTTTCTCCATCACCAGCGCATTTTGGAGCAGAACCTAACTCAAATACAAATGTTATCGACTTAGCATATCCAGGTGTCTTACCAGTTGTTAATAAGCGTGCAGTAGACTGGGCAATGCGTGCTGCAATGGCACTAAATATGGAAATCGCAACAGAATCTAAGTTTGACCGTAAGAACTATTTCTATCCAGATAATCCAAAAGCATATCAAATTTCTCAATTTGATCAACCAATTGGTGAAAATGGATATATCGATATCGAAGTCGACGGTGAAACAAAACGAATCGGTATTACTCGTCTTCACATGGAAGAAGATGCTGGTAAGTCAACACATAAAGGTGAGTATTCATTAGTTGACTTGAACCGTCAAGGTACACCGCTAATTGAAATCGTATCTGAACCAGATATTCGTTCACCTAAAGAAGCATATGCATATTTAGAAAAATTACGTTCAATTATTCAATACACTGGTGTATCAGACGTTAAGATGGAAGAGGGATCTTTACGTTGTGATGCTAACATCTCTTTGCGTCCATATGGTCAAGAAAAATTTGGTACTAAAGCCGAATTGAAAAACTTAAACTCATTTAACTATGTACGTAAAGGTTTAGAATATGAAGAAAAACGCCAAGAAGAAGAATTGTTAAATGGTGGAGAAATCGGACAAGAAACACGTCGATTTGATGAATCTACAGGTAAAACAATTTTAATGCGTGTTAAAGAAGGTTCTGATGATTACCGTTACTTCCCAGAGCCTGACATTGTACCTTTATATATTGATGATGCTTGGAAAGAGCGTGTTCGTCAGACAATTCCTGAATTACCAGATGAGCGTAAGGCTAAGTATGTAAATGAATTAGGTTTACCTGCATACGATGCACACGTATTAACATTGACTAAAGAAATGTCAGATTTCTTTGAATCAACAATTGAACACGGTGCAGATGTTAAATTAACATCTAACTGGTTAATGGGTGGCGTAAACGAATATTTAAATAAAAATCAAGTAGAATTATTAGATACTAAATTAACACCAGAAAATTTAGCAGGTATGATTAAACTTATCGAAGACGGAACAATGAGCAGTAAAATTGCGAAGAAAGTCTTCCCAGAGTTAGCAGCTAAAGGTGGTAATGCTAAACAGATTATGGAAGATAATGGCTTAGTTCAAATTTCTGATGAAGCAACACTTCTAAAATTTGTAAATGAAGCATTAGACAATAACGAACAATCAGTTGAAGATTACAAAAATGGTAAAGGCAAAGCTATGGGCTTCTTAGTTGGTCAAATTATGAAAGCGTCTAAAGGTCAAGCTAATCCACAATTAGTAAATCAACTATTAAAACAAGAATTAGATAAAAGA-(R₂)_(n)-Y (D) Polypeptide sequenceembodiments [SEQ ID NO:2].X-(R₁)_(n)-MHFETVIGLEVHVELKTDSKMPSPSPAHFGAEPNSNTNVIDLAYPGVLPVVNKRAVDWAMRAAMAKNMEIATESKFDRKNYFYPDNPKAYQISQFDQPIGENGYIDIEVDGETKRIGITRLHMEEDAGKSTHKGEYSLVDLNRQGTPLIEIVSEPDIRSPKEAYAYLEKLRSIIQYTGVSDVKMEEGSLRCDANISLRPYGQEKFGTKAELKNLNSFNYVRKGLEYEEKRQEEELLNGGEIGQETRRFDESTGKTILMRVKEGSDDYRYFPEPDIVPLYIDDAWKERVRQTIPELPDERKAKYVNELGLPAYDAHVLTLTKEMSDFFESTIEHGADVKLTSNWLMGGVNEYLNKNQVELLDTKLTPENLAGMIKLIEDGTMSSKIAKKVFPELAAKGGNAKQIMEDNGLVQISDEATLLKFVNEALDNNEQSVEDYKNGKGKAMGFLVGQIMKASKGQANPQLVNQLLKQELDKR-(R₂)_(n)-Y (E)Sequences from Staphylococcus aureus ratB polynucleotide ORF sequence[SEQ ID NO:3].5′-CTGAAAAACTTAAACTCATTTAACTATGTACGTAAAGGTTTAGAATATGAAGAAAAACGCCAAGAAGAAGAATTGTTAAATGGTGGAGAAATCGGACAAGAAACACGTCGATTTGATGAATCTACAGGTAAAACAATTTTAATGCGTGTTAAAGAAGGTTCTGATGATTACCGNTACTTCCCAGAGCCTGACATTGTACCTTTATATATTGATGATGCTTGGAAAGAGCGTGTTCGTCAGACAATTCCTGAATTACCAGATGAGCGTAAGGCTAAGTATGTAAATGAATTACCTTTACTGCATACGATGCNCNCCTATTAACNTIGACTAAAGAAATGTCAGATTTCTTTGAATCAACAATTGGAACNAGGGTGCAGATTGTTAAATTTAACATCTAACTGGGTTAATGGGTGGCGTNAACGAATATTTAAATAAAAATCAANTAGAATTATTAGATACTAAATTAACACCAGAAAATTTAGCAGGTATGATTAAACTTATCGAAGACGGAACAATGAGCAGTAAAATTGCGAAGAAAGTCTTCCCAGAGTTAGGCAGCTAAAGGGTGGGTAATGCTAAACAGATTATGGGANGATAATGGCTNAGTTCAAATTTCTTGATGGAAGCAACAATCTTCTAAAATTGGGTANATGGAAGCATTAGACAAATAACCAACAATCNGTGGAAGATTACAAAAATGGTAAAGGCAAAGCTATGGGGCTTCTTAGTTGGTCAAATTATGAAAGCGTCTAAAGGTCAAGCTAATCCACAATTAGTAAATCAACTATTAAAACAAGAATTAGATAAAAGATAATTTANATCATCAAACTATGAAGATTTAAAAAATAAACCCTTGATTGCTGACTTAGATGCAATCGAGGGTTTATTTATATCTATAGAAGTCATATTACTTTTAACTTTATTCATTGNACATGTTAATGGTAAAAATATTAATTTTATTAATGCGTTAGCTTTAATTATATTAAGGCAAACTGTATAATAAAAAGGTATAAAACATTTGTGTATAAAGACAACATTATATTTACAACATCATTTTAAAGGTAAAATAGCATAACTGACGAAGTCTATATAATGAAGAACGGCAAAAAATGCTGAATAAATAACAAGCTTTGTACATATTGAGATAGTATTTGTTTAAGATACAAGTTGGTCTTTAACGATATTAAGAATGATGAAATAAGACTGAGCCTGGGTCATAAATTCAATGTCCTAGGCACTACAATGTTAATATTGGCAGTAGTTGACTGAAAGAAAATACGCTTGTAACAAGCTTNNNTCAATTCTAGTGGGGCCCCAACATAGAAGCTGACTTTCTGTCAGCTTACAATAATGTGCAAGTNGGGGTGGGGCCCCAAACAAAGAGAATTTCGAAAGGAAATTCTACAGACAATGCAAGTTGGGGTAGAACGAAATAAATTTTGTTAAATATTATTTCTGTCCCACTCCCTATTAGACGAAACAAAGATGAAGTCAAAATATATGAATTTTAAGTAGAAGGATAAGATATGAACAAACGTGCTAGAATCATTTATAACCCGACATCAGGTAAAGAGCTAATTTAAAAGAGAATTACCTGATGCCTTAATAAAATTAGAAAAAGCGGGATATGAAACGAGTGCATATGCAACCGAGAAAATAGGTGATGCCACACTTGAAGCAGAAAGAGCTATGCATGAAAATTATGATGTATTAATCGCTGCAGGTGGTGATGGAACATTAAATGAAGTAGTTAATGGTATCGCAGAAAAGCCTAATCGTCCTAAGCTAGGTGTCATTCCTATGGGTACTGTTAATGACTTTGGACGTGCATTGCATATACCTAATGACATCATGGGGGCACTTGATGTCATCATTGAAGGTCATTCTACTAAAGTAGATATTGGTAAAATGAATAATCGATACTTTATTAATTTAGCTGCAGGCGGACAATTGACGCAAGTCTCTTATGAAACACCGAGTAAATTGAAATCTATTGTTGGTCCATTTGCTTATTACATCAAAGGTTTCGAAATGTTACCTCAAATGAAAGCTGTAGATTTAAGAATTGAATATGATGGTAATGTTTNCCAAGGAGAAGCATTATTATTCTTTNTAGGCTTAACAAATCCAATGGCAGGATTCGAAAAATTAGTGCCCGGACGCTAAGTTAGATGACGGCTATTTCTACGTTNAATNTATAG-3′ (F) RatB polypeptidesequence deduced from the polynucleotide ORF sequence in this table [SEQID NO:4]. NH₂-MKNLNSFNYVRKGLEYEEKRQEEELLNGGEIGQETRRFDESTGKTILMRVKEGSDDYRYFPEPDIVPLYIDDAWKERVRQTIPELPDERKAKYVNELGLLHTMXXY-COOH

[0051] Deposited Materials

[0052] A deposit containing a Staphylococcus aureus WCUH 29 strain hasbeen deposited with the National Collections of Industrial and MarineBacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Sep. 11, 1995 and assigned NCIMB Deposit No. 40771, andreferred to as Staphylococcus aureus WCUH29 on deposit TheStaphylococcus aureus strain deposit is referred to herein as “thedeposited strain” or as “the DNA of the deposited strain.”

[0053] The deposited strain contains the full length ratB gene. Thesequence of the polynucleotides contained in the deposited strain, aswell as the amino acid sequence of the polypeptide encoded thereby, arecontrolling in the event of any conflict with any description ofsequences herein.

[0054] The deposit of the deposited strain has been made under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

[0055] A license may be required to make, use or sell the depositedstrain, and compounds derived therefrom, and no such license is herebygranted.

[0056] Polypeptides

[0057] The polypeptides of the invention include the polypeptide ofTable 1 [SEQ ID NO:2] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of ratB, and also those which have at least 70% identity to apolypeptide of Table 1 [SEQ ID NOS:2 and 4] or the relevant portion,preferably at least 80% identity to a polypeptide of Table 1 [SEQ IDNOS:2 and 4], and more preferably at least 90% similarity (morepreferably at least 90% identity) to a polypeptide of Table 1 [SEQ IDNOS:2 and 4] and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to a polypeptide of Table 1 [SEQID NOS:2 and 4] and also include portions of such polypeptides with suchportion of the polypeptide generally containing at least 30 amino acidsand more preferably at least 50 amino acids.

[0058] The invention also includes polypeptides of the formula set forthin Table 1 (D) [SEQ ID NO:2] wherein, at the amino terminus, X ishydrogen, and at the carboxyl terminus. Y is hydrogen or a metal, R₁ andR₂ is any amino acid residue, and n is an integer between 1 and 1000 Anystretch of amino acid residues denoted by either R group, where R isgreater than 1, may be either a heteropolymer or a homopolymer,preferably a heteropolymer.

[0059] A fragment is a variant polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the aformentioned polypeptides. As with ratB polypeptides fragmentsmay be “free-standing,” or comprised within a larger polypeptide ofwhich they form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

[0060] Preferred fragments include, for example, truncation polypeptideshaving a portion of an amino acid sequence of Table 1 [SEQ ID NOS:2 and4], or of variants thereof, such as a continuous series of residues thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus. Degradation forms of the polypeptides ofthe invention in a host cell, particularly a Staphylococcus aureus, arealso preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

[0061] Also preferred are biologically active fragments which are thosefragments that mediate activities of ratB, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Staphylococcusaureus or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

[0062] Variants that are fragments of the polypeptides of the inventionmay be employed for producing the corresponding full-length polypeptideby peptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

[0063] RatB polypeptides of the invention can interact with ratC and/orratA polypeptides comprising a polypeptide sequence set forth in Table2, to form ratB:ratA and ratC:ratB heterodimers or ratA:ratB:ratChetertrimers. Such heterotrimers and heterotrimers are useful in themethods of the invention, particularly vaccine and drug screeningmethods set forth herein.

[0064] Polynucleotides

[0065] Another aspect of the invention relates to isolatedpolynucleotides, including the full length gene, that encode the ratBpolypeptide having a deduced amino acid sequence of Table 1 [SEQ IDNOS:2 and 4] and polynucleotides closely related thereto and variantsthereof.

[0066] Using the information provided herein, such as a polynucleotidesequence set out in Table 1 [SEQ ID NOS:1 and 3], a polynucleotide ofthe invention encoding ratB polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH29 cells as starting material, followed by obtaining a full lengthclone. For example, to obtain a polynucleotide sequence of theinvention, such as a sequence given in Table 1 [SEQ ID NOS:1 and 3],typically a library of clones of chromosomal DNA of Staphylococcusaureus WCUH 29 in E. coli or some other suitable host is probed with aradiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently, such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Illustrative of the invention, the polynucleotide setout in Table 1 [SEQ ID NO:1] was discovered in a DNA library derivedfrom Staphylococcus aureus WCUH 29.

[0067] The DNA sequence set out in Table 1 [SEQ ID NOS:1] contains anopen reading frame encoding a protein having about the number of aminoacid residues set forth in Table 1 [SEQ ID NOS:2] with a deducedmolecular weight that can be calculated using amino acid residuemolecular weight values well known in the art. The start codon of theDNA in Table 1 is nucleotide number 1 and last codon that encodes anamino acid is number 1425, the stop codon being the next codon followingthis last codon encoding an amino acid.

[0068] RatB of the invention is structurally related to other proteinsof the rat family, as shown by the results of sequencing the DNAencoding ratB of the deposited strain. The protein exhibits greatesthomology to Bacillus subtilis PET112-like protein, encoded by part ofthe DNA sequence from the GenBank deposition with accession numberU49790 protein among known proteins. RatB of Table 1 [SEQ ID NO:2] hasabout 93% identity over its entire length and about 96% similarity overits entire length with the amino acid sequence of Bacillus subtilisPET112-like protein, encoded by part of the DAN sequence from theGenBank deposition with accession number U49790 polypeptide.

[0069] The invention provides a polynucleotide sequence identical overits entire length to the coding sequence in Table 1 [SEQ ID NO:1]. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro-protein sequence. The polynucleotidemay also contain non-coding sequences, including for example, but notlimited to non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences, termination signals, ribosome binding sites,sequences that stabilize mRNA, introns, polyadenylation signals, andadditional coding sequence which encode additional amino acids. Forexample, a marker sequence that facilitates purification of the fusedpolypeptide can be encoded. In certain embodiments of the invention, themarker sequence is a hexa-histidine peptide, as provided in the pQEvector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad.Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al., Cell 37: 767(1984). Polynucleotides of the invention also include, but are notlimited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

[0070] A preferred embodiment of the invention is the polynucleotide ofcomprising nucleotide 1 to 1425 set forth in SEQ ID NO:1 of Table 1which encodes the ratB polypeptide.

[0071] The invention also includes polynucleotides of the formula setforth in Table 1 (C)[SEQ ID NO:1] wherein, at the 5′ end of themolecule, X is hydrogen, and at the 3′ end of the molecule, Y ishydrogen or a metal, R₁ and R₂ is any nucleic acid residue, and n is aninteger between 1 and 1000. Any stretch of nucleic acid residues denotedby either R group, where R is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer.

[0072] The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureus ratB havingthe amino acid sequence set out in Table 1 [SEQ ID NO:2]. The term alsoencompasses polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypeptide (for example, interruptedby integrated phage or an insertion sequence or editing) together withadditional regions, that also may contain coding and/or non-codingsequences.

[0073] The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of Table 1 [SEQ ID NO:2]. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

[0074] Further particularly preferred embodiments are polynucleotidesencoding ratB variants, that have the amino acid sequence of ratBpolypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted oradded, in any combination. Especially preferred among these are silentsubstitutions, additions and deletions, that do not alter the propertiesand activities of ratB.

[0075] Further preferred embodiments of the invention arepolynucleotides that are at least 70% identical over their entire lengthto a polynucleotide encoding ratB polypeptide having an amino acidsequence set out in Table 1 [SEQ ID NOS:2 and 4], and polynucleotidesthat are complementary to such polynucleotides. Alternatively, mosthighly preferred are polynucleotides that comprise a region that is atleast 80% identical over its entire length to a polynucleotide encodingratB polypeptide of the deposited strain and polynucleotidescomplementary thereto. In this regard, polynucleotides at least 90%identical over their entire length to the same are particularlypreferred, and among these particularly preferred polynucleotides, thosewith at least 95% are especially preferred. Furthermore, those with atleast 97% are highly preferred among those with at least 95%, and amongthese those with at least 98% and at least 99% are particularly highlypreferred, with at least 99% being the more preferred.

[0076] Preferred embodiments are polynucleotides that encodepolypeptides that retain substantially the same biological function oractivity as the mature polypeptide encoded by the DNA of Table 1 [SEQ IDNO:1].

[0077] A further preferred embodiment of the invention provides apolynucleotide sequence comprising a ratB polynucleotide sequence andratC polynucleotide set forth in Table 2 [SEQ ID NO:3]. Anotherpreferred embodiment of the invention provides a polynucleotide sequencecomprising ratB polynucleotide sequence and ratA polynucleotide setforth in Table 2 [SEQ ID NO:5]. Yet another preferred embodiment of theinvention provides a polynucleotide sequence comprising the ratBpolynucleotide sequence, the ratA polynucleotide sequence set forth inTable 2 [SEQ ID NO:5] and the ratC set polynucleotide sequence forth inTable 2 [SEQ ID NO:3].

[0078] The invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions” mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in0.1×SSC at about 65° C. Hybridization and wash conditions are well knownand exemplified in Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularlyChapter 11 therein.

[0079] The invention also provides a polynucleotide consistingessentially of a polynucleotide sequence obtainable by screening anappropriate library containing the complete gene for a polynucleotidesequence set forth in SEQ ID NO:1 or SEQ ID NO:3 under stringenthybridization conditions with a probe having the sequence of saidpolynucleotide sequence set forth in SEQ ID NO:1 or a fragment thereof;and isolating said DNA sequence. Fragments useful for obtaining such apolynucleotide include, for example, probes and primers describedelsewhere herein.

[0080] As discussed additionally herein regarding polynucleotide assaysof the invention, for instance, polynucleotides of the invention asdiscussed above, may be used as a hybridization probe for RNA, cDNA andgenomic DNA to isolate full-length cDNAs and genomic clones encodingratB and to isolate cDNA and genomic clones of other genes that have ahigh sequence similarity to the ratB gene. Such probes generally willcomprise at least 15 bases. Preferably, such probes will have at least30 bases and may have at least 50 bases. Particularly preferred probeswill have at least 30 bases and will have 50 bases or less.

[0081] For example, the coding region of the ratB gene may be isolatedby screening using the known DNA sequence provided in SEQ ID NO: 1 tosynthesize an oligonucleotide probe. A labeled oligonucleotide having asequence complementary to that of a gene of the invention is then usedto screen a library of cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

[0082] The polynucleotides and polypeptides of the invention may beemployed, for example, as research reagents and materials for discoveryof treatments of and diagnostics for disease, particularly humandisease, as further discussed herein relating to polynucleotide assays.

[0083] Polynucleotides of the invention that are oligonucleotidesderived from the sequences of SEQ ID NOS:1 and/or 2 may be used in theprocesses herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

[0084] The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

[0085] A precursor protein, having the mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. When prosequences are removed such inactive precursorsgenerally are activated. Some or all of the prosequences may be removedbefore activation. Generally, such precursors are called proproteins.

[0086] In sum, a polynucleotide of the invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

[0087] Vectors, Host Cells, Expression

[0088] The invention also relates to vectors that comprise apolynucleotide or polynucleotides of the invention, host cells that aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

[0089] For recombinant production, host cells can be geneticallyengineered to incorporate expression systems or portions thereof orpolynucleotides of the invention. Introduction of a polynucleotide intothe host cell can be effected by methods described in many standardlaboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULARBIOLOGY, (1986) and Sambrook et al, MOLECULAR CLONING: A LABORATORYMANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989), such as, calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction and infection.

[0090] Representative examples of appropriate hosts include bacterialcells, such as streptococci, staphylococci, enterococci E. coli,streptomyces and Bacillus subtilis cells; fungal cells, such as yeastcells and Aspergillus cells; insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3,BHK, 293 and Bowes melanoma cells; and plant cells.

[0091] A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

[0092] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

[0093] Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

[0094] Diagnostic Assays

[0095] This invention is also related to the use of the ratBpolynucleotides of the invention for use as diagnostic reagents.Detection of ratB in a eukaryote, particularly a mammal, and especiallya human, will provide a diagnostic method for diagnosis of a disease.Eukaryotes (herein also “individual(s)”), particularly mammals, andespecially humans, infected with an organism comprising the ratB genemay be detected at the nucleic acid level by a variety of techniques.

[0096] Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled ratB polynucleotide sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNasedigestion or by differences in melting temperatures. DNA sequencedifferences may also be detected by alterations in the electrophoreticmobility of the DNA fragments in gels, with or without denaturingagents, or by direct DNA sequencing. See, e.g., Myers et al., Science,230: 1242 (1985). Sequence changes at specific locations also may berevealed by nuclease protection assays, such as RNase and S1 protectionor a chemical cleavage method. See, e.g., Cotton et al., Proc. Natl.Acad. Sci., USA. 85: 4397-4401 (1985).

[0097] Cells carrying mutations or polymorphisms in the gene of theinvention may also be detected at the DNA level by a variety oftechniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations. It is particularly preferred to usedRT-PCR in conjunction with automated detection systems, such as, forexample, GeneScan. RNA or cDNA may also be used for the same purpose,PCR or RT-PCR. As an example, PCR primers complementary to a nucleicacid encoding ratB can be used to identify and analyze mutations.Examples of representative primers are shown below in Table 2. Theinvention further provides these primers with 1, 2, 3 or 4 nucleotidesremoved from the 5′ and/or the 3′ end. These primers may be used for,among other things, amplifying ratB DNA isolated from a sample derivedfrom an individual. The primers may be used to amplify the gene isolatedfrom an infected individual such that the gene may then be subject tovarious techniques for elucidation of the DNA sequence. In this way,mutations in the DNA sequence may be detected and used to diagnoseinfection and to serotype and/or classify the infectious agent.

[0098] The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStaphylococcus aureus, and most preferably disease, such as, infectionsof the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.,empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic absces,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and perinephric absces, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyelitis), comprising determining from a sample derivedfrom an individual a increased level of expression of polynucleotidehaving the sequence of Table 1 [SEQ ID NO: 1]. Increased or decreasedexpression of ratB polynucleotide can be measured using any on of themethods well known in the art for the quantation of polynucleotides,such as, for example, amplification, PCR, RT-PCR, RNase protection,Northern blotting and other hybridization methods.

[0099] In addition, a diagnostic assay in accordance with the inventionfor detecting over-expression of ratB protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a ratBprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

[0100] Antibodies

[0101] The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. “Antibodies” as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

[0102] Antibodies generated against the polypeptides of the inventioncan be obtained by administering the polypeptides or epitope-bearingfragments, analogues or cells to an animal, preferably a nonhuman, usingroutine protocols. For preparation of nomoclonal antibodies, anytechnique known in the art that provides antibodies produced bycontinuous cell line cultures can be used. Examples include varioustechniques, such as those in Kohler, G. and Milstein, C., Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole etal., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc. (1985).

[0103] Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

[0104] Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-ratB or from naive libraries (McCafferty,J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology, 10, 779-783). The affinity of these antibodies can alsobe improved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

[0105] If two antigen binding domains are present each domain may bedirected against a different epitope—termed ‘bispecific’ antibodies.

[0106] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptides to purify the polypeptidesby affinity chromatography.

[0107] Thus, among others, antibodies against ratB- polypeptide may beemployed to treat infections, particularly bacterial infections andespecially disease, such as, infections of the upper respiratory tract(e.g., otitis media, bacterial tracheitis, acute epiglottitis,thyroiditis), lower respiratory (e.g., empycma, lung abscess), cardiac(e.g., infective endocarditis), gastrointestinal (e.g., secretorydiarrhoea, splenic absces, retroperitoneal abscess), CNS (e.g., cerebralabscess), eye (e.g., blepharitis, conjunctivitis, keratitis,endophthalmitis, preseptal and orbital cellulitis, darcryocystitis),kidney and urinary tract (e.g. epididymitis, intrarenal and perinephricabsces, toxic shock syndrome), skin (e.g., impetigo, folliculitis,cutaneous abscesses, cellulitis, wound infection, bacterial myositis)bone and joint (e.g., septic arthritis, osteomyelitis).

[0108] Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term “antigenically equivalent derivative” as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm “immunologically equivalent derivative” as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

[0109] The polypeptide, such as an antigenically or immunologicallyequivalent derivative or a fusion protein thereof is used as an antigento immunize a mouse or other animal such as a rat or chicken. The fusionprotein may provide stability to the polypeptide. The antigen may beassociated, for example by conjugation, with an immunogenic carrierprotein for example bovine serum albumin (BSA) or keyhole limpethaemocyanin (KLH). Alternatively a multiple antigenic peptide comprisingmultiple copies of the protein or polypeptide, or an antigenically orimmunologically equivalent polypeptide thereof may be sufficientlyantigenic to improve immunogenicity so as to obviate the use of acarrier.

[0110] Preferably, the antibody or variant thereof is modified to makeit less immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be “humanized”; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

[0111] The use of a polynucleotide of the invention in geneticimmunization will preferably employ a suitable delivery method such asdirect injection of plasmid DNA into muscles (Wolff et al., Hum MolGenet 1992, 1-363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419),delivery of DNA complexed with specific protein carriers (Wu et al., JBiol Chem. 1989: 264,16985), coprecipitation of DNA with calciumphosphate (Benvenisty & Reshef, PNAS, 1986:83,9551), encapsulation ofDNA in various forms of liposomes (Kaneda et al., Science 1989:243,375),particle bombardment (Tang et al., Nature 1992, 356:152, Eisenbraun etal., DNA Cell Biol 1993, 12:791) and in vivo infection using clonedretroviral vectors (Seeger et al., PNAS 1984:81,5849).

[0112] Antagonists and Agonists—Assays and Molecules

[0113] Polypeptides of the invention may also be used to assess thebinding of small molecule substrates and ligands in, for example, cells,cell-free preparations, chemical libraries, and natural productmixtures. These substrates and ligands may be natural substrates andligands or may be structural or functional mimetics. See, e.g., Coliganet al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

[0114] The invention also provides a method of screening compounds toidentify those which enhance (agonist) or block (antagonist) the actionof ratB polypeptides or polynucleotides, particularly those compoundsthat are bacteriostatic and/or bacteriocidal. The method of screeningmay involve high-throughput techniques. For example, to screen foragonists or antagonists, a synthetic reaction mix, a cellularcompartment, such as a membrane, cell envelope or cell wall, or apreparation of any thereof, comprising ratB polypeptide and/or ratCpolypeptide [SEQ ID NO:6] and/or ratA polypeptide [SEQ ID NO:8] and alabeled substrate or ligand of such polypeptide is incubated in theabsence or the presence of a candidate molecule that may be a ratBagonist or antagonist. The ability of the candidate molecule to agonizeor antagonize the ratB polypeptide is reflected in decreased binding ofthe labeled ligand or decreased production of product from suchsubstrate. Molecules that bind gratuitously, i.e., without inducing theeffects of ratB polypeptide are most likely to be good antagonists.Molecules that bind well and increase the rate of product productionfrom substrate are agonists. Detection of the rate or level ofproduction of product from substrate may be enhanced by using a reportersystem. Reporter systems that may be useful in this regard include butare not limited to colorimetric labeled substrate converted intoproduct, a reporter gene that is responsive to changes in ratBpolynucleotide or polypeptide activity, and binding assays known in theart. TABLE 2 Polynucleotide sequence of rat C [SEQ ID NO:5] 5′-ATGACAAAAGTAACACGTGAAGAAGTTGAGCATATCGCGAATCTTGCAAGACTTCAAATTTCTCCTGAAGAAACGGAAGAAATGGCCAACACATTAGAAAGCATTTTAGATTTTGCAAAACAAAATGATAGCGCTGATACAGAAGGCGTTGAACCTACATATCACGTTTTAGATTTACAAAACGTTTTACGTGAAGATAAAGCAATTAAAGGTATTCCGCAAGAATTAGCTTTGAAAAATGCCAAAGAAACAGAAGATGGACAATTTAAAGTGCCTACAATCATGAATGAGGAGGACGCG-3′ Polypeptide sequence of rat C deduced from thesequence of SEQ ID NO:3. [SEQ ID NO:6]MTKVTREEVEHIANLARLQISPEETEEMANTLESILDFAKQNDSADTEGVEPTYHVLDLQNVLREDKAIKGIPQELALKNAKETEDGQFKVPTIMNEEDA-COOH Polynucleotide sequence of ratA [SEQID NO:7] 5′-ATGAGCATTCGCTACGAATCGGTTGAGAATTTATTAACTTTAATAAAAGACAAAAAAATCAAACCATCTGATGTTGTTAAAGATATATATGATGCAATTGAAGAGACTGATCCAACAATTAAGTCTTTTCTAGCGCTGGATAAAGAAAATGCAATCAAAAAAGCGCAAGAATTGGATGAATTACAAGCAAAAGATCAAATGGATGGCAAATTATTTGGTATTCCAATGGGTATAAAAGATAACATTATTACAAACGGATTAGAAACAACATGTGCAAGTAAAATGTTAGAAGGTTTTGTGCCAATTTACGAATCTACTGTAATGGAAAAACTACATAAAGAGAATGCCGTTTTAATCGGTAAATTAAATATGGATGAGTTTGCAATGGGTGGTTCAACAGAAACATCTTATTTCAAAAAAACAGTTAACCCATTTGACCATAAAGCAGTACCAGGTGGTTCATCAGGTGGATCTGCAGCAGCAGTTGCAGCTGGCTTAGTACCATTTAGCTTAGGTTCAGACACAGGTGGTTCAATTAGACAACCGGCTGCATATTGTGGCGTTGTCGGTATGAAACCAACATACGGTCGTGTATCTCGATTTGGATTAGTTGCTTTTGCATCTTCATTAGACCAAATTGGTCCATTGACTCGAAATGTAAAAGATAATGCAATCGTATTAGAAGCTATTTCTCCTCCACATGTTAATGACTCTACAAGTGCACCAGTTGATGATGTAGACTTTACATCTGAAATTGGTAAAGATATTAAAGGATTAAAAGTTGCATTACCTAAAGAATACTTAGGTGAAGGTGTAGCTGATGACGTAAAAGAAGCAGTTCAAAACGCTGTAGAAACTTTAAAATCTTTACCTGCTGTCGTTGAGGAAGTATCATTGCCAAATACTAAATTTGGTATTCCATCATATTACGTGATTGCATCATCAGAAGCTTCGTCAAACCTTTCTCGTTTTGACGGAATTCGTTATGGTTATCATTCTAAAGAAGCTCATTCATTAGAAGAATTATATAAAATGTCAAGATCTGAAGGTTTCGGTAAAGAAGTAAAACGTCGTATTTTCTTAGGTACATTTGCATTAAGTTCAGGTTACTACGATGCTTACTATAAAAAATCTCAAAAAGTTAGAACATTGATTAAAAATCACTTTGATAAAGTATTCGAAAATTATGATGTAGTAGTTGGTCCAACAGCGCCTACAACTGCGTTTAATTTAGGTGAAGAAATTGATGATCCATTAACAATGTATGCCAATGATTTATTAACAACACCAGTAAACTTAGCTGGATTACCTGGTATTTCTGTTCCTTGTGGACAATCAAATGGCCGACCAATCGGTTTACAGTTCATTGGTAAACCATTCGATGAAAAAACGTTATATCGTGTCGCTTATCAATATGAAACACAATACAATTTACATGACGTTTATGAAAAATTA-3′ Polypeptidesequence of ratA deduced from the sequence of SEQ ID NO:5. [SEQ ID NO:8]NH₂-MSIRYESVENLLTLIKDKKIKPSDVVKDIYDAIEETDPTIKSFLALDKENAIKKAQELDELQAKDQMDGKLFGIPMGIKDNIITNGLETTCASKMLEGFVPIYESTVMEKLHKENAVLIGKLNMDEFAMGGSTETSYFKKTVNPFDHKAVPGGSSGGSAAAVAAGLVPFSLGSDTGGSIRQPAAYCGVVGMKPTYGRVSRFGLVAFASSLDQIGPLTRNVKDNAIVLEAISGADVNDSTSAPVDDVDFTSEIGKDIKGLKVALPKEYLGECVADDVKEAVQNAVETLKSLGAVVEEVSLPNTKFGIPSYYVIASSEASSNLSRPDGIRYGYHSKEAHSLEELYKMSRSEGFGKEVKRRIFLGTPALSSGYYDAYYKKSQKVRTLIKNDFDKVFENTDVVVGPTAPTTAFNLGEEIDDPLTMYANDLLTTPVNLAGLPGISVPCGQSNGRPIGLQFIGKPFDEKTLYRVAYQYETQYNLhDVYEKL-COOH

[0115] Another example of an assay for ratB antagonists is a competitiveassay that combines ratB and/or ratC [SEQ ID NO:5 or 6] and/or ratA [SEQID NO:7 or 8] and a potential antagonist with ratB-binding molecules,recombinant ratB binding molecules, natural substrates or ligands, orsubstrate or ligand mimetics, under appropriate conditions for acompetitive inhibition assay. ratB can be labeled, such as byradioactivity or a colorimetric compound, such that the number of ratBmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

[0116] Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing ratB-induced activities, thereby preventing the action of ratBby excluding ratB polypeptide and/or ratC polypeptide [SEQ ID NO:6]and/or ratA polypeptide [SEQ ID NO:8] from binding.

[0117] Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1990); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of ratB.

[0118] Each of the DNA sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the DNA sequences encoding the aminoterminal regions of the encoded protein or Shine-Delgarno or othertranslation facilitating sequences of the respective mRNA can be used toconstruct antisense sequences to control the expression of the codingsequence of interest.

[0119] The invention also provides the use of the polypeptide,polynucleotide or inhibitor of the invention to interfere with theinitial physical interaction between a pathogen and mammalian hostresponsible for sequelae of infection. In particular the molecules ofthe invention may be used: in the prevention of adhesion of bacteria, inparticular gram positive bacteria, to mammalian extracellular matrixproteins on in-dwelling devices or to extracellular matrix proteins inwounds; to block ratB protein-mediated mammalian cell invasion by, forexample, initiating phosphorylation of mammalian tyrosine kinases(Rosenshine et al., Infect. Immun. 60:2211 (1992); to block bacterialadhesion between mammalian extracellular matrix proteins and bacterialratB proteins that mediate tissue damage and; to block the normalprogression of pathogenesis in infections initiated other than by theimplantation of in-dwelling devices or by other surgical techniques.

[0120] The antagonists and agonists of the invention may be employed,for instance, to inhibit and treat disease, such as, infections of theupper respiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empycma, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

[0121] Vaccines

[0122] Another aspect of the invention relates to a method for inducingan immunological response in an individual, particularly a mammal whichcomprises inoculating the individual with ratB, or a fragment or variantthereof, adequate to produce antibody and/or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Staphylococcus aureus infection. Also provided aremethods whereby such immunological response slows bacterial replication.Yet another aspect of the invention relates to a method of inducingimmunological response in an individual which comprises delivering tosuch individual a nucleic acid vector to direct expression of ratB, or afragment or a variant thereof, for expressing ratB, or a fragment or avariant thereof in vivo in order to induce an immunological response,such as, to produce antibody and/or T cell immune response, including,for example, cytokine-producing T cells or cytotoxic T cells, to protectsaid individual from disease, whether that disease is alreadyestablished within the individual or not. One way of administering thegene is by accelerating it into the desired cells as a coating onparticles or otherwise.

[0123] Such nucleic acid vector may comprise DNA, RNA, a modifiednucleic acid, or a DNA/RNA hybrid.

[0124] A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within rt an immunological response, induces an immunologicalresponse in such individual to a ratB or protein coded therefrom,wherein the composition comprises a recombinant ratB or protein codedtherefrom comprising DNA which codes for and expresses an antigen ofsaid ratB or protein coded therefrom. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity or cellular immunity such as that arising from CTL orCD4+ T cells.

[0125] A ratB polypeptide or a fragment thereof may be fused withco-protein which may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

[0126] Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

[0127] Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStaphylococcus aureus infection, in mammals, particularly humans.

[0128] The polypeptide may be used as an antigen for vaccination of ahost to produce specific antibodies which protect against invasion ofbacteria, for example by blocking adherence of bacteria to damagedtissue. Examples of tissue damage include wounds in skin or connectivetissue caused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

[0129] The invention also includes a vaccine formulation which comprisesan immunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual, and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

[0130] While the invention has been described with reference to certainratB protein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

[0131] Compositions, Kits and Administration

[0132] The invention also relates to compositions comprising thepolynucleotide or the polypeptides discussed above or their agonists orantagonists. The polypeptides of the invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa polypeptide of the invention and a pharmaceutically acceptable carrieror excipient. Such carriers may include, but are not limited to saline,buffered saline, dextrose, water, glycerol, ethanol and combinationsthereof. The formulation should suit the mode of administration. Theinvention further relates to diagnostic and pharmaceutical packs andkits comprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

[0133] Polypeptides and other compounds of the invention may be employedalone or in conjunction with other compounds, such as therapeuticcompounds.

[0134] The pharmaceutical compositions may be administered in anyeffective, convenient manner including, for instance, administration bytopical, oral, anal, vaginal, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes amongothers.

[0135] In therapy or as a prophylactic, the active agent may beadministered to an individual as an injectable composition, for exampleas a sterile aqueous dispersion, preferably isotonic.

[0136] Alternatively the composition may be formulated for topicalapplication for example in the form of ointments, creams, lotions, eyeointments, eye drops, ear drops, mouthwash, impregnated dressings andsutures and aerosols, and may contain appropriate conventionaladditives, including, for example, preservatives, solvents to assistdrug penetration, and emollients in ointments and creams. Such topicalformulations may also contain compatible conventional carriers, forexample cream or ointment bases, and ethanol or oleyl alcohol forlotions. Such carriers may constitute from about 1% to about 98% byweight of the formulation; more usually they will constitute up to about80% by weight of the formulation.

[0137] For administration to mammals, and particularly humans, it isexpected that the daily dosage level of the active agent will be from0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The physician in anyevent will determine the actual dosage which will be most suitable foran individual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

[0138] In-dwelling devices include surgical implants, prosthetic devicesand catheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

[0139] The composition of the invention may be administered by injectionto achieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphylococcus aureus wound infections.

[0140] Many orthopaedic surgeons consider that humans with prostheticjoints should be considered for antibiotic prophylaxis before dentaltreatment that could produce a bacteremia. Late deep infection is aserious complication sometimes leading to loss of the prosthetic jointand is accompanied by significant morbidity and mortality. It maytherefore be possible to extend the use of the active agent as areplacement for prophylactic antibiotics in this situation.

[0141] In addition to the therapy described above, the compositions ofthis invention may be used generally as a wound treatment agent toprevent adhesion of bacteria to matrix proteins exposed in wound tissueand for prophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

[0142] Alternatively, the composition of the invention may be used tobathe an indwelling device immediately before insertion. The activeagent will preferably be present at a concentration of 1 μg/ml to 10mg/ml for bathing of wounds or indwelling devices.

[0143] A vaccine composition is conveniently in injectable form.Conventional adjuvants may be employed to enhance the immune response. Asuitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, andsuch dose is preferably administered 1-3 times and with an interval of1-3 weeks. With the indicated dose range, no adverse toxicologicaleffects will be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

[0144] Each reference disclosed herein is incorporated by referenceherein in its entirety. Any patent application to which this applicationclaims priority is also incorporated by reference herein in itsentirety.

EXAMPLES

[0145] The examples below are carried out using standard techniques,which are well known and routine to those of skill in the art, exceptwhere otherwise described in detail. The examples are illustrative, butdo not limit the invention.

Example 1 Strain Selection, Library Production and Sequencing

[0146] The polynucleotide having the DNA sequence given in SEQ ID NO:1was obtained from a library of clones of chromosomal DNA ofStaphylococcus aureus in E. coli. The sequencing data from two or moreclones containing overlapping Staphylococcus aureus DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO:1. Libraries may beprepared by routine methods, for example:

[0147] Methods 1 and 2 below.

[0148] Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

[0149] Method 1

[0150] Total cellular DNA is mechanically sheared by passage through aneedle in order to size-fractionate according to standard procedures.DNA fragments of up to 11 kbp in size are rendered blunt by treatmentwith exonuclease and DNA polymerase, and EcoRI linkers added. Fragmentsare ligated into the vector Lambda ZapII that has been cut with EcoRI,the library packaged by standard procedures and E. coli infected withthe packaged library. The library is amplified by standard procedures.

[0151] Method 2

[0152] Total cellular DNA is partially hydrolyzed with a one or acombination of restriction enzymes appropriate to generate a series offragments for cloning into library vectors (e.g., RsaI, PalI, Alul,Bshl235I), and such fragments are size-fractionated according tostandard procedures. EcoRI linkers are ligated to the DNA and thefragments then ligated into the vector Lambda ZapII that have been cutwith EcoRI, the library packaged by standard procedures, and E. coliinfected with the packaged library. The library is amplified by standardprocedures.

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide sequence selected from the group consisting of: (a) apolynucleotide having at least a 70% identity to a polynucleotideencoding a polypeptide comprising the amino acid sequence of SEQ IDNO:2; (b) a polynucleotide which is complementary to the polynucleotideof (a); (c) a polynucleotide having at least a 70% identity to apolynucleotide encoding the same mature polypeptide expressed by theratB gene contained in the Staphylococcus aureus of the depositedstrain; and (d) a polynucleotide comprising at least 15 sequential basesof the polynucleotide of (a), (b) or (c).
 2. The polynucleotide of claim1 wherein the polynucleotide is DNA.
 3. The polynucleotide of claim 1wherein the polynucleotide is RNA.
 4. The polynucleotide of claim 2comprising the nucleic acid sequence set forth in SEQ ID NO:1.
 5. Thepolynucleotide of claim 2 comprising nucleotide 1 to 1425 set forth inSEQ ID NO:1.
 6. The polynucleotide of claim 2 which encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:2.
 7. Avector comprising the polynucleotide of claim
 1. 8. A host cellcomprising the vector of claim
 7. 9. A process for producing apolypeptide comprising: expressing from the host cell of claim 8 apolypeptide encoded by said DNA.
 10. A process for producing a ratBpolypeptide or fragment comprising culturing a host of claim 8 underconditions sufficient for the production of said polypeptide orfragment.
 11. A polypeptide comprising an amino acid sequence which isat least 70% identical to the amino acid sequence of SEQ ID NO:2.
 12. Apolypeptide comprising an amino acid sequence as set forth in SEQ IDNO:2.
 13. An antibody against the polypeptide of claim
 11. 14. Anantagonist which inhibits the activity or expression of the polypeptideof claim
 11. 15. A method for the treatment of an individual in need ofratB polypeptide comprising: administering to the individual atherapeutically effective amount of the polypeptide of claim
 11. 16. Amethod for the treatment of an individual having need to inhibit ratBpolypeptide comprising: administering to the individual atherapeutically effective amount of the antagonist of claim
 14. 17. Aprocess for diagnosing a disease related to expression or activity ofthe polypeptide of claim 11 in an individual comprising: (a) determininga nucleic acid sequence encoding said polypeptide, and/or (b) analyzingfor the presence or amount of said polypeptide in a sample derived fromthe individual.
 18. A method for identifying compounds which interactwith and inhibit or activate an activity of the polypeptide of claim 11comprising: contacting a composition comprising the polypeptide with thecompound to be screened under conditions to permit interaction betweenthe compound and the polypeptide to assess the interaction of acompound, such interaction being associated with a second componentcapable of providing a detectable signal in response to the interactionof the polypeptide with the compound; and determining whether thecompound interacts with and activates or inhibits an activity of thepolypeptide by detecting the presence or absence of a signal generatedfrom the interaction of the compound with the polypeptide.
 19. A methodfor inducing an immunological response in a mammal which comprisesinoculating the mammal with ratB polypeptide of claim 11, or a fragmentor variant thereof, adequate to produce antibody and/or T cell immuneresponse to protect said animal from disease.
 20. A method of inducingimmunological response in a mammal which comprises delivering a nucleicacid vector to direct expression of ratB polypeptide of claim 11, orfragment or a variant thereof, for expressing said ratB polypeptide, ora fragment or a variant thereof in vivo in order to induce animmunological response to produce antibody and/or T cell immune responseto protect said animal from disease.